1. Field of the Invention
The present invention relates to a method for collecting N-terminus of protein that is useful in determining protein amino acid sequence.
2. Disclosure of the Related Art
One way to determine the amino acid sequence of a protein is by protein sequencers. In sequencing a protein using a typical protein sequencer, an N-terminus labeled protein is subjected to Edman method to cleave and remove the N-terminal amino acid one at a time. The removed amino acid is converted to a more stable form. The obtained end product is then detected by UV and determined based on the retention time in high performance liquid chromatography (HPLC). In this manner, the amino acid sequence of a protein can be determined sequentially from its N-terminus. However, the protein sequencing using protein sequencers has several drawbacks. First, Edman method cannot be applied to any protein with modified N-terminus, resulting in failure of directly sequencing. Although the protein with modified N-terminus can be sequenced indirectly by first making it into fragments, such an approach only permits the determination of the internal sequence of the protein, but not the sequencing from the N-terminus. Second, in Edman method, the side reactions caused by the low stability of the end product and the like often make HPLC analysis difficult. Third, the low UV-absorbance of the end product of Edman method can lead to low sensitivity.
In recent years, mass spectrometers with extremely high sensitivity and accuracy have been developed and the data obtained by mass spectroscopy analysis alone can permit effective, reliable determination of partial amino acid sequences of a peptide and what protein a peptide originates from. For this reason, mass spectroscopy has become a major tool in protein analysis. In mass spectroscopy-based protein analysis, a protein is cleaved into peptide fragments by enzymatically digestion and the like, and masses of the peptide fragments are measured. By comparing this obtained data with the sequence data stored in a database, the identity of the protein is determined. Among such mass spectroscopic techniques are peptide mass fingerprinting (PMF) (See, for example, Henzel W J., Billeci T M., Stults J T., Wong S C., Grimley C., and Watanabe C. “Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.” Proceedings of the National Academy of Sciences of the United States of America 90 (1993): 5011–5015, and, James P., Quadroni M., Carafoli E., and Gonnet G. “Protein identification by mass profile finger printing.” Biochemical Biophysical Research Communications 195 (1993): 58–64), and, MS/MS analysis for directly obtaining the information concerning the amino acid sequence of a peptide (See, for example, Hunt D F., Yates J R., Shabanowitz J., Winston S., and Hauer C H. “Protein sequencing by tandem mass spectrometry” Proceedings of the National Academy of Sciences of the United States of America 83 (1986): 6233–6237). However, none of these techniques are capable of distinguishing between the N-terminal peptide fragment and the other peptide fragments of a protein. Therefore, these techniques only allow the determination of internal amino acid sequences of a protein, but not the sequencing from the N-terminus of a protein.